AMINO ACID COMPOSITION AND NUTRITIVE

VALUE OF THE ALGA SPZRULZNA MAXIMA

By G. CLfiMENT, C. GIDDEY and R. MENZI

Spirulina maxima is an alga, rich in organic nitrogenous constituents, used for food in the Chad Republic.
Amino acids, vitamins, and nutritive value were determined for a strain of the algae grown in an open-air
pilot production unit. The dried alga, more than 60% of which was proteinaceous material, contained
all the essential amino acids in fairly good concentration except that the amount of the sulphur amino
acids was low. Nutrition experiments on rats showed that the biological value of the alga was acceptable and
was correlated with the chemical score (protein index). Dried Sgirrrlina maxima also contained several
vitamins, particularly p-carotene (pro-vitamin A).

Introduction The wells allow COz injection to be made and ensure circula-
In the northern part of the Chad Republic there are shallow tion of the aqueous growth medium by convection. The
stretches of water from which phytoplankton has been aqueous medium contains the required mineral salts. Most
collected for food from time immemorial by tribes living in of the COPused in the experimental programme was pure,
relative isolation. The predominant species of phyto- some was obtained by combustion from a standard oil burner.
plankton is Spirulina maxima, a blue-green alga.* S. The alga was collected by filtration, sun-dried and finely
maxima varies in its morphology, but is characterised by a milled (hammer mill). Production was estimated to reach
spiral structure. Most of the units have seven spirals and a 4 M 5 t of dried algae/hectare/year. The powder was mixed,
length of 230-300 p (Fig. 1). The adsorption spectra of the
pigments show the presence of chlorophyll, carotenoid,
phycoerythrin, and phycocyanin (Fig. 2). The brackish
water in which the plant grows is alkaline (pH 9.5-10.0) and
has a high salt content (24 g/l) mainly owing to sodium
carbonate and bicarbonate (HCO,, 11010 mg/l; C 0 i 2 ,
5029 mg/l).
The algae are collected during April to October by the
women and spread on the sand to be sun-dried. The dried
cake, named die, is sold mainly on the local markets. Die
is used in a sauce prepared and used daily as a dressing for the
basic local dish, the biri?a thick sauce with millet. Accord-
ing to local opinion, die advantageously replaces meat sauce
and largely contributes to maintaining the nutritional value of
the diet when meat is scarce. At one time, die was the main
source of protein of the tribes. The dried alga contains
about 62 % proteinaceous matter (Kjeldahl N x 6.25),
2-3 % lipids, 16-18 % carbohydrates, and mineral salts.**
A strain of S. maxima has been isolated in the laboratory
and grown in an open-air pilot production unit in the south of
France using a synthetic medium composed of mineral salts
and C02.2

Experimental
Production and sampling
The production unit consists of a pool, 20 X 4.5 m in size
and 0.07m deep, joined at each end to two wells 1.10 m deep.

* Division: Cyanophyta; Order: Nostocales; Family: Oscilla-

toriaceae FIG.1. Spirulina maxima
** Unpublished results by the authors Magnification 160 X

J. Sci. Fd Agric., 1967, Vol. 18, November

498 Clkment et al. : Amino Acid Composition and Nutritive Value of the Alga Spirulina maxima
I I I I I and the hydrochloric acid was eliminated under vacuum.
02c \ - I
01 I I I
350 L50 550 650 750
WAVELENGTH, m p
FIG.2. Absorption spectrum of suspension of Spirulina maxima in
its growth medium
and the samples for analysis were collected according to the
usual 'Square sampling technique'. The nitrogen content
was 8.9% (Kjeldahl)* and the residual moisture was 9.3%
(1lo", 24 h).
Vitamins
Analyses have been made on the milled dried algae accord-
ing to the methods given by Strohecker & Henning.3
,%Carotene,Photometric analysis
B1 (thiamine), Fluorometric analysis
B2 (riboflavine, lactoflavine), Fluorometric analysis
Bg (pyridoxine), Microbiological analysis (Saccharomyces
klausbergensis and S . carlsbergensis)
312 (LLD factor), Microbiological analysis (Auchromonas
malhamensis)
C, Photometric analysis with dinitrophenylhydrazine.
Protein precipitated at iso-electric point
The dried algae powder was extracted for 2 hours by hot
water (SO"), solid/liquid ratio 1 : 10. The original pH was
adjusted to 10 by N NaOH solution before the extraction.
When the extraction was over, the insoluble residue was
removed by centrifugation and the centrifugate was acidified
to pH 4 with 10% hydrochloric acid. The iso-electrically
precipitated proteins were recovered by centrifugation and
freeze-dried. The nitrogen recovered during the extraction
was 62% of the original nitrogen of the algae. The protein
material obtained contained a relatively large quantity of non-
protein compounds. The nitrogen content was 10.2%
(Kjeldahl N) and the moisture content was 9 -72%.
Protein hydrolysis
The samples (algae powder or iso-electrically precipitated
protein), 50 mg were hydrolysed by 6 N-HC~ (50 ml) in sealed
tubes at llO"for24 or 48 hours. Thehydrolysateswere filtered,
* Catalyser of Dumazert & Mafcelet (Bull. SOC. chim. biol., 1938,
20, 201) supplied by Prolabo, Paris
The moist residue (addition of a small amount of water during
the evaporation) was quantitatively transferred in 50 ml
buffer (pH 2 . 2 ) .
Amino acid analysis
The hydrolysates were analysed with the Unichrom
Beckman ion exchange chromatographic analyser (ninhydrin
reaction, 570 mp and 440 mp optical density recording). The
respective concentrations of the amino acids were calculated
by the half base x height method.
Tryptophan analysis
Tryptophan was determined quantitatively by the Portner
& Hoegl4 method. The hydrolysis was performed with
papain.
Determination of biological value (BV), net protein utilisation
(NPU) and digestibility (D)
The methods of were used. Some modification
was required for calculating the digestibility and the biological
value.
Rats (strain Wistar WAG) from 4 litters of 8 rats each
were systematically distributed into 8 groups of 4 rats (males
and females indiscriminately). The groups were 25 days old.
From the 25th to the 32nd day the animals were acclimatised
and fed a standard diet. From the 32nd to the 42nd day they
were fed the test diets. They were killed on the 42nd day.
During the experiments, they were weighed every day and
their excrement was collected. The faeces were separated
from the urine by sheets of filter paper fitted under the cages.
Faeces and filter papers were stored in a freezer before
analysis.
Carcass analysis
The dead rats were dehydrated in an oven at 105" for 48 h.
Each carcass was crushed in a mortar and milled with a
hammer mill. The resulting powder was autoclaved for
90 minutes with 600 ml 20% HC1 at 151". The slurry was
filtered. The nitrogen content of the solution was analysed
by the Kjeldahl method.
Urine and faeces analyses
The filter papers were hydrolysed as above with 500 ml
HC1 (20%). The faeces were hydrolysed as above with
200 ml HCl(20 %).
The nitrogen contents of the faeces, and the filter papers
containing the urine were measured by Kjeldahl analyses.
Diets
Diets of casein, raw Spirulina maxima and stewed S.
maxima (in 20% water for 1 h on a boiling-water bath) were
assessed with reference to a non-protein diet.
The composition of the basic diet included: 150 g mar-
garine, 100 g potato starch, 150 g glucose, 20 g vitamin con-
centrate,* 30 g maize starch, 50 g mineral salt mixture,**
500 g rice starch.
* Vitamin diet fortification mixture in dextrose; Nutritional
Biochemical Corp., Cleveland 28, Ohio, U.S.A.
** Hawk Oser Salt mixture No. 3. NBC, Cleveland
J. Sci. Fd Agric., 1967, Vol. 18, November
 Climent et al. : Amino Acid Composition and Nutritive Value of the Alga Spirulina maxima 499

For the other diets, a part of the rice starch was replaced by Protein chemical score
the protein component in such a way as to include 10% of Table I11 compares the respective concentrations of the
pure protein (N x 6.25) in the total composition. The essential amino acids of the provisional pattern10 of several
feeds were granulated before use. characteristic proteins and of Spirulina maxima. The
chemical scores were calculated. The limiting amino acid is
underlined for each protein. For S. maxima, according to
Results and Discussion the chemical analysis, all the essential amino acids are
sufficient in general with the exception of the sulphur amino
Vitamins acids.
Table I summarises the results. The chemical score was calculated according to the recom-
mendation of an F.A.O. report:l0
The dried alga is thus especially rich in vitamin A. % sulphur acid Spirulina maxima
Chemical score = x 100
% sulphur amino acid of the
Amino acid composition provisional pattern
- 1.80 2.37
Table I1 summarises the composition of the various -~ x 100 = 43% or 7x 100 = 5 6 % if the maxi-
4.20 4 20
preparations . mum possible content of sulphur amino acid is accepted.
Tryptophan and lysine, which are deficient in many
TABLE
I protein foods, are present in sufficient concentration in S.
Vitamin content of Spirulina maxima grown in open air on synthetic maxima.
media, filtered on sieves, and sun-dried
(9.5 % moisture basis) Biological value (BV)
The biological value was calculated, as indicated by
Concentration Bender,5-9from the nitrogen value of the ingested food, of the
Vitamin mg/100 g alga u.l. rats bodies, and of the excrement (urine and faeces) and
metabolic loss. In addition digestibility (D) was measured.
/J-Carotene (pro-vitamin A) 22.5 37,500 The net protein utilisation (NPU) was calculated from the
B1 (thiamine, aneurine) 1.38 460 equation :
BZ(riboflavin, lactoflavine) 2.85 - NPU = BV x D .
B6 (pyridoxine) 0.131 - The weight curves of rats fed with the diets of casein, and
Bl2 (LLD factor, cyanocobalamine) 25.5 pg -
C 10.3 200
* limiting amino acid
TABLE
11
Amino acid composition of Spirulina maxima
(g of amino acid/l6 g N)

d e f Standard
Amino acids a b C
deviation

Alanine 5.99 6.05 7.45 7.32 7.15 6.8

Arginine 6.83 6.07 7.0 5.7 6.55 6.85 6.5
Aspartic acid 8.68 8.0 9.0 8.45 9.0 8.6
Cystine 0.55 0.24 i-Gi6-i
Glutamic acid 13.87 12.3 14.3 12.00 10.3 12.6
Glycine 4.49 4.35 4.9 4.55 5.3 4.75
Histidine 1,86 1.55 1.7 1.55 2.01 1.91
Isoleucine 6.05-6.0 5.55-5.55 5.9 5.82-5.92 6.48-7.05 0.51
Leucine 7.57-8.2 7.0 -7.56 8.65 7 . 5 -8.55 7.90-9.25 0.70
Lysine 5.034.8 5.154.6 5.22 4.624.26 4.27-3.90 4.444.22 4.59 0.43
Methionine 1.29-1.34 1.65-1 - 5 2.74 1,084.97 0.46 1.30 1.37 0.35
Phenylalanine 4 . 9 2 4 . 72 4.434.07 4.7 5.40-5.22 5.95-5.64 0.63
Proline 4.13 3.68 3.7 4.04 4.05 3.90
Serine 4.63 3.68 4.55 4.05 4.05 4.20
Threonine 5.0 4 . 9 5 4.284.20 4.73 4.464.38 4.40-4.68 4.56 0.10
Tryptophan 1.18 1.40
Tyrosine 4.23-3.6 3 68-3 48 4.35 4.18-3.4 4.184.49 0.43
Valine 6.3 -6.4 6.15 1.38 6.35-6.4 6.98-6.90 6.49 0.39
NH3 1.84 1.31 0.26 1.08 1.69 1.24

a, b and c Crude S . maxima, 24 h hydrolysis

d Crude S. maxima 24 h hydrolysis; over-dosage (3 x ) for controlling the concentration of some amino acids
e lso-electrically precipitated protein, 24 h hydrolysis
f lso-electrically precipitated protein, 48 h hydrolysis
Mean value ( a ) was calculated for each amino acid
Standard deviation was calculated only for the essential amino acids
J. Sci. Fd Agric., 1967, Vol. 18, November
500 Clkment et al. : Amino Acid Composition and Nutritive Value of the Alga Spirulina maxima
TABLE
111
Essential amino acids in Spirulina maxima and other typical protein foods
(g of amino acid/l6 g N)

Amino acids Provisional Proteins Spirulina

pattern" milk egg beef soya meal groundnut meal maxima

Isoleucine 4.2 6.4 6.8 5.2 5.3 4.1 6.03

Leucine 4.8 9.9 9.0 8.0 7.7 6.0 8.02
Lysine 4.2 7.8 6.3 8.4 6.3 3.5 4.59
Phenylalanine 2.8 4.9 6.0 4.0 4.95 5.0 4.97
Tyrosine 2.8 5.1 4.4 3.3 3.2 3.6 3.95
Sulphur amino acids
(total) 4.2 3.3
~ 5.4 3.7 3.15 2.4 1.80
1.37
~

Methionine 2.2 2.4 3.1 2.4 0.9 1.37

Threonine 2.8 4.6 5.0 4.3 3.95 2.7 4.56
Tryptophan 1.4 1.4 1-7 I .2
- 1.49 1.1 1.40
Valine 4.2 6.9 7.4 5.5 3.45 4.9 6.49
Chemical score 100 78 100 83 73 54 43-56

raw and stewed S . maxima are shown in Figs 3 , 4 and 5. The

reference curves (diet without proteins) are shown on each
respective graph.
Table IV summarises the net protein utilisation, the
digestibility, the biological value, and the protein chemical
score of S . maxima calculated from nutrition and analytical
experiments .
A relatively good correlation was found between the
chemical score calculated from standard amino acid analyses
and the NPU determined on rats. The limiting amino acid
assessed analytically is methionine ; however, another
essential amino acid, present but not in an available form,
might contribute to reducing the protein value.
Artificially produced dried S . maxima, which contains TIME, d a y s
more than 60% of proteinaceous material of good digestibility
and has been a constituent of the diet of African tribes for FIG.4. Weight of rats fed on diet containing raw Spirulina maxima
centuries, could thus become a valuable food protein basis if I, control group, non-protein diet; 11, 111, S. maxima
supplemented with amino acids or mixed with products, e.g.
cereals, containing complementary proteins.

i I OO
I
2 L
1 I
6
I
8
I
10
1

30
0 2 L 6 8 10 T I M E , days
TIME, days
FIG.5. Weight of rats fed on Spirulina maxima diets
FIG.3. Weight of rats fed on casein diet I, 11, control groups, non-protein diet
111, IV, raw S. maxima
I, control group, non-protein diet; 11, 111, IV, casein diet V. VI, stewed S. maxima

J. Sci. Fd Agric., 1967, Vol. 18, November

 Maleki & Sarkissian: Canning and Storage Efects on Lebanese Orange Juices 50 1

TABLEIV Acknowledgment
Nutritive value of Spirulina maxima The authors wish to thank Dr. Blanc, of the Institut Suisse
des Vitamines, Lausanne, for his contribution and Dr. J.
Net protein Digestibility Biological Chemical Rossi, Geneva, for his useful suggestions and discussions.
Dried alga utilisation value score
Institut Battelle,
7 route de Drize,
Raw 45.6-49.8 75.5-76.7 60-65 43-56
(6 experiments) Geneva, Switzerland
Stewed 36.0-40.0 74.3 48-54 43-56 and
(4 experiments) Institut FranGais d u Petrole,
97 Rueil Malmaison,
France
Received 24 February, 1967
Amended manuscript received 27 April, 1967

References
1. Geitler, L., Kryptogamen-Flora, Cyanophyceae von Europa, 4. Portner, I. I., & Hoegl, I. I., Analytica chim. Acm, 1953, 8,
1932, 14 (Leipzig : Akademische Verlagsgesellschaft) 29
5. Bender, A. E., Br. J. Nutr., 1956, 10, 135
2. C1ement3 G . , M.7 Lk Trambouze9 P.y 7th 6. Miller, D. S., & Bender, A. E., Br. J . Nutr., 1955, 9, 382
Petroleum Congress, Mexico, 1967, PD 37; R ~ P PInst.. Fr- 7. Bender, A. E., & ~ ~B. H.,
~ B ~1J .. N , ~ ~1957,
~ , 11,
, 140
PPtrole No. 13778, 1966 8. Bender, A. E., Proc. Nutr. Soc., 1965, 24 (2), 190
3. Strohecker, I. I., & Henning, I. I., Vitaminbestimmungen, 9. Bender, A. E., Proc. Nutr. Soc., 1958, 17 (I), 85
1963 (Weinheim : Verlag Chemie) 10. h d e s de nutrition de la F A 0 No. 16, Rome, 1958

EFFECT OF CANNING AND STORAGE ON THE

CHEMICAL COMPOSITION AND ORGANOLEPTIC
QUALITY OF JUICES OF DIFFERENT VARIETIES
OF ORANGES GROWN IN LEBANON
By M. MALEKI* and S. SARKISSIAN

Juice from five different varieties of oranges grown in Lebanon was canned according to commercial
practices and stored at room temperature. At weekly intervals for seven weeks, chemical analyses for
ascorbic acid, total acidity, total soluble solids, nitrogen, ash, total lipids, total solids, essential oils and
flavonoids were performed on each sample.
The fresh juice from the Blood and Washington Navel varieties compared favourably in chemical
composition with a concentrated frozen product from the U.S.A. ;the juice from Blood withstood canning
and storage better than all other varieties tested. In all samples, there was a significant decrease in the
levels of ascorbic acid and of essential oils during storage.
Statistical analysis of organoleptic evaluations showed that, in general, blends of two or three different
varieties were preferred to the juice from a single variety.

Introduction There is n o published work on the chemical composition

The Mediterranean Sea coast is one of the worlds major
citrus growing regions; the total production in this area
during the 19641965 harvesting season was estimated a t
5,300,000 tons, of which 145,000 tons were from Leban0n.l
* Present address : Faculty of Agriculture, Pahlavi University,
Shiraz, Iran
J. Sci. Fd Agric., 1967, Vol. 18, November
or organoleptic properties of the juice of the different varieties
of oranges grown in Lebanon. Moreover, studies o n single-
strain orange juice from other regions are few and there are
no reports of a complete chemical analysis of any variety.
There have been extensive investigations of the effect of
storage on ascorbic acid and o n other individual chemical
components of orange juice. In a study of browning, Joslyn2